KR20190024984A - Production of activated Clostridium neurotoxin - Google Patents

Abstract

The present invention relates to a method for producing an activated Clostridial neurotoxin essentially free of inactivated products, to compositions comprising such, and to its use in therapy.

Description

Production of activated Clostridium neurotoxin

The present invention relates to a method for producing an activated Clostridial neurotoxin essentially free of inactivated products, to compositions comprising such, and to its use in therapy.

Bacteria in the genus Clostridia produce very potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. An example of such a clostridial toxin is seed. By C. tetani (TeNT) and seeds. Botulinum (C. botulinum) (BoNT) serotypes A neurotoxin produced by the AG, as well as seeds. C. baratii and seeds. And those produced by C. butyricum .

Some of the most potent toxins among Clostridium neurotoxins are known. By way of example, botulinum neurotoxin has a median lethal dose (LD ₅₀ ) value for mice ranging from 0.5 to 5 ng / kg, depending on the serotype. Both tetanus and botulinum toxin act by inhibiting the function of the affected neurons, specifically the release of neurotransmitters. The botulinum toxin acts in the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, whereas the tetanus toxin acts in the central nervous system.

In fact, the Clostridium neurotoxin is synthesized as a single-chain polypeptide, which is posttranslationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage often occurs at specific cleavage sites, referred to as activation sites, which are located between cysteine residues that provide interspecific disulfide bonds. The most active form of neurotoxin is this two-chain form. The two strands are termed the heavy chain (H-chain) having a molecular mass of approximately 100 kDa and the light chain (L-chain) having a molecular mass of approximately 50 kDa. The H-chain includes an N-terminal potential component (H _N domain) and a C-terminal targeting component (H _C domain). The cleavage site is located between the L-chain and the potential domain component. After binding of the H _C domain to its target neuron and internalization of the bound toxin through the endosome into the cell, the H _N domain translocates the L-chain into the endosomal membrane and into the cytosol, and the L- (Also known as non-cytotoxic proteases).

Non-cytotoxic proteases function by proteolytic cleavage of intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or Syntaxin) - see Gerald K (2002) Cell & Molecular Biology " (4th edition) by John Wiley & Acronym SNARE term soluble NSF attachment receptor; - derived from (S oluble N SF A ttachment Re ceptor where NSF is N- ethyl maleimide meaning a sensitivity factor (N -ethylmaleimide- S ensitive F actor) ). SNARE protein is essential for intracellular vesicle fusion and is therefore essential for the secretion of molecules through vesicle transport from cells. The protease function is zinc-dependent endopeptidase activity and exhibits high substrate specificity for the SNARE protein. Thus, once delivered to an intended target cell, the non-cytotoxic protease can inhibit cell secretion from the target cell. The L-chain protease of Clostridium neurotoxin is a non-cytotoxic protease that cleaves the SNARE protein.

In view of the parasitic nature of the SNARE protein, Clostridium neurotoxins such as botulinum toxins have been used successfully in a wide range of therapies.

By way of example, the present inventors refer to William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004), which inhibits neuronal transmission in a number of therapies and cosmetic or esthetic applications to Clostridium neurotoxin, for example botulinum neurotoxin (BoNT), BoNT / A, BoNT / B, BoNT / C 1, BoNT / D, BoNT / E, BoNT / F and BoNT / G, and Te tanuseu neurotoxin for (TeNT) - for example, a commercial botulinum toxin product is currently used to treat a variety of conditions including but not limited to focal stiffness, superior stiffness, lower stiffness, neck anomalies, eyelid spasm, unilateral facial spasm, axillary hyperhidrosis, chronic migraine, Activity, nasal wrinkles, and severe external facial wrinkles. To treat neuromuscular disorders (see US 6,872,397); For treating uterine disorders (see US 2004/0175399); For treating ulcers and gastroesophageal reflux disease (see US 2004/0086531); For treating anxiety disorders (see US 6,319,505); For treating eye disorders (see US 2004/0234532); For treating the bladder axis (see US 2004/0151740); For treating strabismus (see US 2004/0126396); For treating pain (see US 6,869,610, US 6,641,820, US 6,464,986 and US 6,113,915); For treating fibromyalgia (see US 6,623,742, US 2004/0062776); For treating lower back pain (see US 2004/0037852); For treating muscle damage (see US 6,423,319); For treating the same headache (see US 6,838,434); For treating tension headaches (see US 6,776,992); For treating headaches (see US 6,458,365); For the reduction of migraine headache pain (see US 5,714,469); For treating cardiovascular diseases (see US 6,767, 544); For the treatment of neurological disorders such as Parkinson's disease (see US 6,620,415, US 6,306,403); For treating neuropsychiatric disorders (see US 2004/0180061, US 2003/0211121); For treating endocrine disorders (see US 6,827,931); For treating thyroid disorders (see US 6,740,321); For treating cholinergic affective disorders (see US 6,683,049); For treating diabetes (see US 6,337, 075, US 6,416, 765); For treating pancreatic disorders (see US 6,261,572, US 6,143, 306); For the treatment of cancer such as bone tumors (see US 6,565,870, US 6,368,605, US 6,139,845, US 2005/0031648); For the treatment of ear disorders (see US 6,358,926, US 6,265,379); For treating autonomic disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunctions (see US 5,437,291); For the treatment of skin lesions associated with skin cell-proliferative disorders (see US 5,670,484); For the management of neurogenic inflammatory disorders (see US 6,063,768); For reducing hair loss and stimulating hair growth (see US 6,299,893); For treating mouth sag (see US 6,358,917); For reducing appetite (see US 2004/40253274); For dentistry and procedures (see US 2004/0115139); For treating neuromuscular disorders and conditions (see US 2002/0010138); For treating various disorders and conditions and associated pain (see US 2004/0013692); For treating conditions due to mucus hypersecretion, such as asthma and COPD (see WO 00/10598); And Clostridium neurotoxic therapy for treating non-neuronal conditions such as inflammation, endocrine, exocrine, immunological, cardiovascular, bone conditions (see WO 01/21213). All of the above disclosures are incorporated herein by reference in their entirety.

The use of non-cytotoxic proteases such as Clostridium neurotoxins (e. G., BoNT and TeNT) in the therapeutic and cosmetic treatment of humans and other mammals provides an ongoing widespread range Of the disease and disease.

Traditionally, the production of the Clostridial neurotoxin has been reported to be by the seed. Followed by incubation of the botulinum bacteria followed by isolation and purification of Clostridium neurotoxin complex. However, Mr. Botulinum is a spore forming bacterium and therefore requires specialized incubation equipment and facilities not required for the cultivation of bacteria such as Escherichia coli ( E. coli ). Thus, the increased use of Clostridium neurotoxin leads to the need for alternative and / or improved methods of producing and purifying the Clostridial neurotoxin.

Clostridial neurotoxin is initially expressed as a single chain polypeptide. To be fully active, the single chain form should be converted to a two-chain form, which requires proteolytic cleavage at the site located between the light and heavy chains (the " activation loop "). In vitro activation of the Clostridial neurotoxin can be achieved by the addition of a suitable protease. However, it is known in the related art that, prior to complete activation, unwanted proteolytic activity occurs at sites other than the activated loop frequently resulting in the formation of undesired "truncated" (or "hyperactive") products It is a recurring problem. The formation of such products is particularly problematic when the activated two-chain Clostridium neurotoxin is intended for use as a pharmaceutical product requiring a highly pure and functional preparation.

An open solution to the problem requires sequence modification of some form of the BoNT amino acid sequence; Examples include inserting a particular protease recognition site within the activation loop and thereby altering its sequence.

For example, WO0114570 describes a recombinant nucleic acid molecule encoding a BoNT protein. However, the nucleic acid molecule of WO0114570 is modified to replace the natural cleavage site with a non-natural cleavage site. Thus, the method of WO0114570 also teaches that insertion of non-natural cleavage sites is required for optimal BoNT expression.

US20080103098. Chain recombinant BoNT protein comprising the expression of a recombinant nucleic acid construct in E. coli host cells. However, this method requires the insertion of a specific non-natural (i.e., non-clostridium) pentapeptide sequence into the loop domain of the neurotoxin. The inserted pentapeptide sequence is endogenous upon cell lysis. To form an activated cleavage site that is cleaved by a coli protease. Thus, the method of US20080103098 teaches that BoNT sequences should be modified by insertion of non-natural cleavage sites to achieve optimal BoNT expression.

WO2010094463 describes a process for separating processed BoNT from unprocessed or partially processed BoNT requiring the use of antibodies against BoNTs that are not proteolytically processed and / or partially processed.

Another approach described in WO2012020057 is based on inserting modified linkers that impart physicochemical properties between light and heavy chains flanked by protease cleavage sites. The physicochemical properties of the linker should allow it to isolate, for example, a BoNT that is partially processed or unprocessed from the processed (activated) BoNT.

Thus, there is a need in the art for a method of producing an essentially pure biologically active agent of the full-length activated Clostridium neurotoxin without relying on the insertion of an exogenous sequence. The present invention solves this problem by providing a novel method as specified in the claims, which modifies the Clostridial neurotoxin amino acid sequence or avoids the requirement of using a purification tag.

Summary of the Invention

In accordance with a first aspect, the present invention provides a method for treating a single-stranded Clostridia neurotoxin comprising contacting an activated, single-stranded Clostridium neurotoxin with an activating enzyme until at least 90% of the single-stranded Clostridium neurotoxin polypeptide is converted into a 2- Lt; RTI ID = 0.0 > Clostridium < / RTI > neurotoxin.

In a second aspect, the invention provides an active 2-chain Clostridium neurotoxin obtained by the method according to the invention.

In a third aspect, the invention provides a pharmaceutical composition comprising an active, 2-chain Clostridium neurotoxin according to the invention essentially free of single-chain Clostridium neurotoxin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the preparation of biologically active, full-length, essentially pure Clostridial neurotoxin preparations by a semi-intuitive approach.

An intuitive solution to the problem of producing a preparation of essentially pure Clostridia neurotoxin is to adjust the conditions of the activation process so that a minimal amount of truncated product is formed. However, the inventors have discovered that using such an approach generates a portion of the Clostridial neurotoxin remaining untouched (inactive). In other words, such a process produces a mixture of a full-activated Clostridium neurotoxin product, a full-length inactivated (single chain) Clostridium neurotoxin product, and a truncated Clostridial neurotoxin product. In this regard, it is important to note that both the battle-inactivated product and the truncated activated product are undesirable and much more so when the activated two-chain Clostridium neurotoxin is intended for use as a pharmaceutical product . Thus, such undesired products must be removed during subsequent steps of the manufacturing process.

The present inventors further found that it is extremely difficult to isolate the full-length activated Clostridium neurotoxin (2-chain) from the battle-inactivated Clostridial neurotoxin (single chain).

The present inventors have surprisingly found that it is easier to remove unwanted byproducts (over-activated or truncated product) in a subsequent purification step by causing the activation step to proceed to a later step, essentially free of any battle-deactivated product .

While not intending to be bound by theory, the difficulty encountered with separating the full-length activated Clostridium botulinum toxin from the battle-inactivated botulinum toxin is that the available physicochemical difference between the bat- tynically activated botulinum toxin and the bat- inactivated botulinum toxin is insufficient It is hypothesized that this is because. It is additionally hypothesized that undesired truncated Clostridium neurotoxin byproducts have greater physicochemical differences than activated full-length Clostridial neurotoxins due to changes in the major amino acid sequences between products. These larger physicochemical differences can be used in purification techniques known in the art, such as column chromatography, to achieve essentially pure products that are suitable for achieving separation of the full-activated Clostridia neurotoxin and for use in therapy .

Thus, in a first aspect, there is provided a method of treating a single-stranded Clostridium neurotoxin comprising contacting an activated Clostridium neurotoxin with an activating enzyme until greater than 90% of the single-stranded Clostridium neurotoxin polypeptide is converted to a 2- A method for producing dime neurotoxin is provided.

Preferably, the single-stranded Clostridium neurotoxin is contacted with the activating enzyme until more than 95% of the single-stranded Clostridium neurotoxin polypeptide is converted to the 2-chain form. More preferably, the single-stranded Clostridium neurotoxin comprises greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.9%, or 100% of the single chain Clostridium neurotoxin polypeptide And is contacted with the activating enzyme until it is converted into a 2-chain form.

The term " active Clostridial neurotoxin " as used herein refers to Clostridium neurotoxin capable of binding to and internalizing the target cell and inhibiting SNARE-driven secretion of the neurotransmitter from the cell. It is well known in the art that the level of biological activity of the Clostridial neurotoxin is much higher when the Clostridial neurotoxin is in the 2-chain (or " activated ") form than when it is in the single chain form . Thus, the biologically active Clostridial neurotoxin is preferably a two-chain (or " activated ") Clostridial neurotoxin.

The term " activation " as used herein refers to the conversion of a single-stranded Clostridium neurotoxin polypeptide into a two-chain (or active) form.

As used herein, an " activating enzyme " (or " activating protease ") can be, for example, a Clostridium Means an endopeptidase capable of cleaving a single-stranded Clostridium neurotoxin at a site located between the neurotoxin light and heavy chains (referred to in the art as " activation loop ").

Examples of suitable activating enzymes according to the invention are members of the cysteine, serine and metalloprotease families, such as trypsin, Lys-C, Lys-N and arginylendopeptidase (endoproteinase Arg-C, LeR) As well as active BoNT hydrolases as disclosed in EP 2 524 963 Al, the disclosure of which is incorporated herein by reference.

In a preferred embodiment, the activating enzyme is trypsin.

In a most preferred embodiment, the activation enzyme is small trypsin.

We have found that small trypsin is more suitable for activating botulinum neurotoxin than other trypsin sources, such as pig trypsin. In fact, the present inventors have found that by trypsin, complete activation can be achieved with very little undesired truncated product compared to when pig trypsin is used.

This came as a surprising discovery to the present inventors in view of the fact that the porcine trypsin amino acid sequence is the most commonly used recombinant GMP-grade tryptic sequence and appears to be a reliable source of trypsin for this purpose. It is in fact preferred to use GMP grade, animal-free raw materials for the production of GMP-grade Clostridial neurotoxin intended to be used in therapy.

In a preferred embodiment, the activation enzyme is a GMP-grade enzyme, such as GMP-grade trypsin, more preferably GMP-grade small trypsin, even more preferably GMP-grade recombinant trypticin. &Quot; GMP-grade " means manufactured with quality standards and traceability suitable for use in manufacturing GMP (Good Manufacturing Practice Standards).

In a preferred embodiment, the activation enzyme is a tryptic, such as a native trypsin obtained from a small pancreas (TPCK-treated or non-TPCK treated) or recombinant trypticin.

In accordance with the present invention, the small trypsin is at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, or at least 90% identical to SEQ ID NO: 1 (UniProtKB Accession No. P00760) , 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.

Bovine trypsin is commercially available from and can be obtained from any suitable source, for example, Applied Biotechnology Institute (Applied Biotechnology Institute) ^(® trip binary (TrypZean)). Alternatively, the small trypsin can be obtained by recombinant expression of an amino acid sequence encoding a protein having at least 90% identity with SEQ ID NO: 1.

We also found that the specificity of trypticin for the activation loop of BoNT / E was higher at a pH of about 6-7. This result was unexpected because the performance of trypsin in the related art is well known to be optimal at about pH 8.

In a preferred embodiment, the step of contacting the single-stranded Clostridium neurotoxin with small trypsin is carried out at a pH of from 5 to 7.5, preferably from 6 to 7, for example at a pH of about 6.5.

The step of contacting the single-stranded Clostridia neurotoxin with an activating enzyme, preferably trypsin, is preferably carried out for 10 to 50 hours, preferably 15 to 30 hours, for example about 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours.

The step of contacting the single-stranded Clostridial neurotoxin with trypsin is preferably performed at room temperature (or ambient temperature). The room temperature is typically 16 to 27 占 폚, preferably 18 to 25 占 폚, for example about 18, 19, 20, 21, 22, 23, 24 or 25 占 폚.

The concentration of trypsin is typically 0.5 to 50 μg per mg Clostridium neurotoxin, preferably 1 to 20 μg per mg Clostridium neurotoxin, more preferably 2 to 10 μg per mg Clostridial neurotoxin, For example, about 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg trypsin per mg of Clostridium neurotoxin.

The concentration of trypsin can be expressed in USP (US Pharmacopoeia) units, in which case it is typically between 1 and 500 USP units per mg Clostridium neurotoxin, preferably between 3 and 100 USP per mg Clostridium neurotoxin, More preferably 5 to 50 USP units per mg Clostridium neurotoxin, such as about 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, It is a trypsin of 50 USP units.

According to a preferred embodiment, the step of contacting the single-stranded Clostridium neurotoxin with trypsin is carried out at room temperature at a pH of from 6 to 7, with a concentration of from 1 to 20 [mu] g of cytotoxin per mg of Clostridium neurotoxin and from 15 to 30 hours It is carried out for a duration.

According to another preferred embodiment, the step of contacting the single-stranded Clostridial neurotoxin with trypsin is carried out at room temperature at a pH of from 6 to 7, with a concentration of 3 to 100 USP per gram of Clostridium neurotoxin, To < / RTI > 30 hours.

According to one embodiment of the invention, the method further comprises the step of removing the truncated (hyperactivated) Clostridial neurotoxin, in particular the truncated 2-chain Clostridium neurotoxin.

The term " truncated double-stranded tridinium neurotoxin " (or " unwanted byproduct " or " hyperactivated Clostridial neurotoxin ") refers to single chain botulinum Refers to products resulting from cleavage of neurotoxins.

Such steps may be performed, for example, by contacting the resulting two-chain Clostridium neurotoxin with a suitable chromatographic resin. An example of a suitable chromatographic resin is a mixed mode chromatography resin. An example of a suitable mixed mode chromatography resin is a ceramic hydroxyapatite (CHT) type II 40 micrometer resin (BioRad).

Many different types of Clostridial neurotoxins are suitable for use in the present invention. Thus, in the context of the present invention, the term " Clostridial neurotoxin " (or " Clostridium toxin " Botulinum (botulinum neurotoxin serotypes A, B, C ₁ , D, E, F and G) Tetani (Tetanus neurotoxin), Mr. Butyricum (botulinum neurotoxin serotype E), and seeds. Toxins produced by Baratay (botulinum neurotoxin serotype F), as well as modified Clostridial neurotoxins or derivatives derived from any of the foregoing. The term " Clostridial neurotoxin " also encompasses naturally occurring botulinum neurotoxin hybrids, mosaics and chimeras.

Thus, in one embodiment, the Clostridial neurotoxin of the present invention or for use in the invention is selected from the group consisting of Clostridia botulinum, Clostridia tetani, Clostridia barratii, and Clostridia var. ≪ / RTI > butyricum, and < RTI ID = 0.0 > butyricum. ≪ / RTI >

Botulinum neurotoxin (BoNT) is a large protein complex consisting of BoNT itself complexed to a number of accessory proteins. It is produced by botulinum. And the current of seven different classes of botulinum neurotoxin, that is botulinum neurotoxin serotypes A, B, C _1, D, E, F and G is present, all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished on the basis of inactivation by specific neutralizing anti-sera, with this classification by serotype correlated with percent sequence identity at the amino acid level. The BoNT proteins of a given serotype are further divided into different subtypes based on amino acid percent sequence identity.

Suitably, the Clostridium neurotoxin for use in the invention or of the present invention is a botulinum neurotoxin (BoNT), preferably a BoNT / A, BoNT / B, BoNT / C 1, BoNT / D, BoNT / E , One or more BoNT (s) selected from the group consisting of BoNT / F and BoNT / G.

In one embodiment, the Clostridial neurotoxin can be BoNT / A. The reference BoNT / A sequence has Uniproth KB Accession No. P10845 (SEQ ID NO: 2).

In another embodiment, the Clostridial neurotoxin may be BoNT / B. The reference BoNT / B sequence has Uniproth KB Accession No. P10844 (SEQ ID NO: 3).

In another embodiment, the Clostridial neurotoxin may be BoNT / C. The reference BoNT / C ₁ sequence has Uniproth KB Accession No. P18640 (SEQ ID NO: 4).

In another embodiment, the Clostridial neurotoxin can be BoNT / D. The reference BoNT / D sequence has Uniproth KB Accession No. P19321 (SEQ ID NO: 5).

In another embodiment, the Clostridial neurotoxin can be BoNT / E. The reference BoNT / E sequence has UniParc ID UPI00000010A3 (SEQ ID NO: 6).

In another embodiment, the Clostridial neurotoxin can be BoNT / F. The reference BoNT / F sequence has Uniproth KB accession number YP_001390123 (SEQ ID NO: 7).

In another embodiment, the Clostridial neurotoxin may be BoNT / G. The reference BoNT / G sequence has Uniproth KB Accession No. Q60393 (SEQ ID NO: 8).

In one embodiment, the Clostridial neurotoxin may be TeNT. The reference TeNT sequence has Uniproth KB Accession No. P04958 (SEQ ID NO: 9).

In one embodiment, the Clostridial neurotoxin of the invention or for use in the invention comprises a BoNT / E activation loop. The BONT / E activation loop comprises the amino acid sequence of SEQ ID NO: 10: "CKNIVSVKGIRKSIC" (corresponding to amino acid residues C412 to C426 of SEQ ID NO: 6) or SEQ ID NO: 10 and 1,2,3,4 or 5 amino acid residue insertions , Deletion or substitution of the sequence.

In one embodiment, the BONT / E activation loop has a sequence consisting of SEQ ID NO: 10. In another embodiment, the BONT / E activation loop has a sequence that differs from SEQ ID NO: 10 by one amino acid residue insertion, deletion or substitution. In another embodiment, the BONT / E activation loop has a sequence that differs from SEQ ID NO: 10 by as much as two amino acid residue insertions, deletions or substitutions. In another embodiment, the BONT / E activation loop has a sequence that differs from SEQ ID NO: 10 by as much as three amino acid residue insertions, deletions or substitutions. In another embodiment, the BONT / E activation loop has a sequence that differs from SEQ ID NO: 10 by four amino acid residue insertions, deletions or substitutions. In another embodiment, the BONT / E activation loop has a sequence that differs from SEQ ID NO: 10 by as much as five amino acid residue insertions, deletions or substitutions.

In a preferred embodiment, the Clostridial neurotoxin is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to BoNT / E, %, 99%, or 100% identical amino acid sequence.

The term " Clostridial neurotoxin " is also intended to encompass a modified Clostridial neurotoxin and derivatives thereof, including but not limited to those described below. The modified Clostridium neurotoxin or derivative may contain one or more modified amino acids relative to the native (unmodified) form of the Clostridial neurotoxin, or may comprise a natural (unmodified) form of the Clostridial neurotoxin , Or may contain one or more deleted amino acids relative to the native (unmodified) form of the Clostridial neurotoxin. By way of example, a modified Clostridial neurotoxin may have a modified amino acid sequence in more than one domain relative to a native (unmodified) Clostridial neurotoxin sequence. Such modifications may alter functional aspects of the neurotoxin, such as biological activity or persistence.

The modified Clostridium neurotoxin can have one or more modifications (e.g., a modified H _C domain) in the amino acid sequence of the heavy chain, wherein the modified heavy chain is higher (higher) than the native (unmodified) Clostridium neurotoxin Or to a lower affinity target nerve cell. Such modifications in the H _C domain can be used to identify residues or proteins (SV2 or synaptotagent) binding sites within the ganglioside binding site of the H _C domain that alter the binding of the target neuron to the ganglioside receptor and / Lt; RTI ID = 0.0 > a < / RTI > Examples of such modified Clostridial neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entirety.

The modified Clostridial neurotoxin may have one or more modifications to the amino acid sequence of the light chain, for example, substrate binding or modification in the catalytic domain that can alter or alter the SNARE protein specificity of the modified LC. Examples of such modified Clostridial neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are incorporated herein by reference in their entirety.

Modified Clostridium neurotoxin may comprise one or more modifications that increase or decrease the biological activity and / or biological persistence of the modified Clostridial neurotoxin.

The term " Clostridium neurotoxin " is intended to encompass a chimeric (or hybrid) Clostridial neurotoxin. The chimeric Clostridium neurotoxin comprises at least a portion of a light chain from one Clostridium neurotoxin type or subtype thereof and at least a portion of a heavy chain from another Clostridial neurotoxin type or subtype.

In one embodiment, the chimeric Clostridium neurotoxin may contain the entire light chain from one Clostridium neurotoxin type or subtype and the heavy chain from another Clostridial neurotoxin type or subtype. In another embodiment, the chimeric Clostridium neurotoxin may contain one Clostridium neurotoxin type or sub-type portion of the heavy chain (e. G., A binding domain), and another portion of the heavy chain may contain another Claus Gt; neurotoxin < / RTI > type or subtype. Similarly or alternatively, the therapeutic element may comprise a light chain portion from a different Clostridial neurotoxin type or subtype. Such a hybrid or chimeric Clostridium neurotoxin may, for example, be administered to a patient immunologically tolerant to a given Clostridial neurotoxin subtype with a concentration lower than the average concentration of the receptor for a given Clostridial neurotoxin heavy chain binding domain To a patient who may have a protease-resistant variant of a membrane or vesicle toxin substrate (e. G., SNAP-25, VAMP, . Hybrid and chimeric Clostridium neurotoxins are described in US 8,071,110 and GB 1607901.4 (not yet published), which are incorporated herein by reference in their entirety. Thus, in one embodiment, the Clostridium neurotoxin for purification according to the method or use of the present invention may be a engineered Clostridium neurotoxin, suitably it may be a engineered chimeric Clostridium neurotoxin have.

The term " Clostridial neurotoxin " is intended to encompass the re-targeted Clostridial neurotoxin. In re-targeted Clostridial neurotoxins, the Clostridial neurotoxin is modified to include an exogenous ligand known as the targeting moiety (TM). The TM is selected to provide binding specificity for the target cell of interest and the natural binding portion of the Clostridial neurotoxin (e. G., H _C domain, or H _CC domain) may be removed as part of the re- . Re-targeting techniques are described, for example, in EP-B-0689459; WO 1994/021300; EP-B-0939818; US 6,461,617; US 7,192,596; WO 1998/007864; EP-B-0826051; US 5,989,545; US 6,395,513; US 6,962,703; WO 1996/033273; EP-B-0996468; US 7,052,702; WO 1999/017806; EP-B-1107794; US 6,632,440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192; And WO 1999/58571; All of which are incorporated herein by reference in their entirety. Thus, in one embodiment, engineered Clostridium neurotoxin for use in the present invention may be engineered re-targeted Clostridial neurotoxin.

The present invention also encompasses the use of Clostridium neurotoxin comprising a " destructive cleavage site ". In the Clostridial neurotoxin, the non-native protease cleavage site is incorporated into the Clostridial neurotoxin at a location selected to cause the cleavage at the site to reduce or inactivate the Clostridial neurotoxin activity. The destructive protease cleavage site may be susceptible to cleavage by a local protease in the event that the Clostridial neurotoxin migrates to a non-target location after administration. Suitable non-natural protease cleavage sites include those described above. Clostridial neurotoxins including destructive cleavage sites are described in WO 2010/094905 and WO 2002/044199, both of which are incorporated herein by reference in their entirety.

In a preferred embodiment, the Clostridium neurotoxin of the present invention or for use in the present invention is free of the complexed protein present in the naturally occurring Clostridial neurotoxin complex.

In one embodiment, the Clostridial neurotoxin of the present invention or for use in the present invention comprises a polypeptide sequence set forth as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9, or a polypeptide sequence having at least 65% RTI ID = 0.0 > 70% < / RTI > sequence identity.

In one embodiment, the Clostridial neurotoxin of the present invention or for use in the invention comprises a polypeptide sequence as set forth in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9, 0.0 > 80% < / RTI > sequence identity.

In one embodiment, the Clostridial neurotoxin of the present invention or for use in the invention comprises a polypeptide sequence set forth as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9, RTI ID = 0.0 > 90% < / RTI > sequence identity.

In one embodiment, the Clostridial neurotoxin of the present invention or for use in the invention comprises a polypeptide sequence set forth as SEQ ID NO: 2, 3, 4, 5, 6, 7, 8 or 9, or a polypeptide sequence having at least 95% Or < / RTI > 99% sequence identity.

In a preferred embodiment, the Clostridial neurotoxin of the present invention or for use in the present invention has at least 65%, 70%, 75%, 80%, 85%, 90% identity with the polypeptide sequence shown as SEQ ID NO: %, 95%, or 99% sequence identity.

&Quot; Percent sequence identity " between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequence. Thus, percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. % Sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. Determination of percent identity and percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which will be familiar to those of ordinary skill in the art.

In one embodiment, the single-stranded Clostridium neurotoxin for use in the invention is administered to a host cell, such as a bacterial, insect, yeast, microorganism, mammalian or plant cell, or a single-chain Clostridial neuron Is obtained by expressing the gene encoding the toxin. Preferably, the heterologous host cell is a human. It's Cola.

In another aspect, the invention provides an active 2-chain Clostridium neurotoxin obtainable by a method according to the present invention.

In another aspect, the present invention provides a pharmaceutical composition comprising an active, two-chain Clostridium neurotoxin according to the present invention, essentially free of single-chain Clostridium neurotoxins.

As used herein, the term "essentially pure" or "essentially free" means that the level of undesirable contaminants (or byproducts) is less than 10%, for example, 9%, 8%, 7%, 6% 4%, 3%, 2%, 1%, or less than 0.1%.

In a preferred embodiment, the pharmaceutical compositions according to the invention comprise less than 10%, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% Single chain Clostridium neurotoxin.

In a more preferred embodiment, the pharmaceutical composition according to the invention comprises less than 10%, for example 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% (Hyperactively activated) Clostridial neurotoxin. ≪ / RTI >

In a preferred embodiment, the pharmaceutical composition according to the present invention comprises less than 5% of single-stranded Clostridium neurotoxin and less than 5% of truncated double-stranded Clostridium neurotoxin.

In a more preferred embodiment, the pharmaceutical composition according to the present invention comprises less than 1% of single-stranded Clostridium neurotoxin and less than 1% of truncated double-stranded Clostridium neurotoxin.

In a more preferred embodiment, the pharmaceutical composition according to the present invention comprises less than 0.1% of single-stranded Clostridium neurotoxin and less than 0.1% of truncated double-stranded Clostridium neurotoxin.

The relative amounts of single chain, 2-chain and truncated 2-chain Clostridium neurotoxins can be determined by methods well known in the relevant art, for example by SDS-PAGE followed by density measurement analysis (Size exclusion, ion exchange, reverse-phase, hydrophobic interaction chromatography) for capillary electrophoresis or for the evaluation of purity (for example, see Example 1) Lt; / RTI >

In one embodiment, the pharmaceutical compositions according to the invention are for use in therapy.

A pharmaceutical composition according to the present invention may be used for the treatment of a disease, condition or syndrome selected from muscle disorders, neuromuscular disorders, neurological disorders, ophthalmic disorders, pain disorders, psychological disorders, joint disorders, inflammatory disorders, endocrine disorders and urinary disorders, Can be used to treat or prevent:

· Corneal swelling, nystagmus, esotropia, diplopia, eyelid varus, posterior eyelid retraction, orbital myopathy, obsessive-compulsive disorder, ocular discomfort, Ophthalmic disorders selected from the group consisting of concomitant misalignment, asynchronous misalignment, primary or secondary esotropia or exotropia, intercranial ophthalmoplegia, splinting, Duane's syndrome, and upper eyelid retraction;

• Stiffness, idiopathic focal dystonia, unilateral facial spasm, sagittal, pediatric or adult (eg, in patients with cerebral palsy, post-stroke, multiple sclerosis, traumatic brain injury or spinal cord injury), muscle stiffness, Facial nerve palsy, facial palsy, coincidental ataxia, facial nerve palsy, facial nerve palsy, facial nerve palsy, facial nerve paralysis, arm-and-moving-fingers-syndrome), progression, primary sequelae, spasmodic seizures, stiff-man syndrome, foot palsy, facial palsy, painful-arm-and-moving-fingers-syndrome Dystonic foot inversion, jaw anomalies, Rabbit syndrome, cerebellar tremor, III nerve palsy, dysrhythmias, dyspepsia, Palate, seizures, Extensor truncal dystonia, facial nerve palsy combined movement, secondary dysrhythmias, Parkinson's disease, Huntington's chorea, epilepsy, off-period dystonia, , Head trauma, muscular dystrophy and benign convulsive fibrous spasm syndrome;

· Ear-related vocal disorders, ear disorders, hearing loss, ear clicks, tinnitus, dizziness, Meniere's disease, dementia, stuttering, dysphagia, dysphagia, laryngeal occlusion during chronic aspiration, A deep bite of lip, a malignant melanoma, a malignant melanoma, granuloma, ventricular dystonia, malignant vocal malfunction, mutational malignant malformation, mouth opening disorder, snoring, voice progression, And laryngeal palsy; Otorhinolaryic disorder including First Bite Syndrome;

· Persistent sphincter dysfunction, persistent sphincter of Oddi hypertension, intestinal muscle disorder, pubic symphyseal syndrome, anismus, rheumatoid arthritis, gallbladder, dysphagia, dysphagia, dementia, constipation, temporomandibular joint dysfunction, Gastrointestinal disorders including dysfunction, gastrointestinal or esophageal motility dysfunction, diffuse esophageal spasm and gastroparesis;

Neurogenic bladder dysfunction, obsessive-compulsive bladder, neurogenic detrusor hyperactivity, bladder spasms, urinary incontinence, urinary incontinence, urinary incontinence, Genitourinary disorders including hypertrophic bladder neck, dysuria, interstitial cystitis, vaginal seizure, endometriosis, pelvic pain, enlarged prostate (benign prostatic hyperplasia), prostatitis, prostate cancer and persistent febrile illness;

Skin cell proliferative disorders, skin wounds, psoriasis, rose intolerance, acne; Rare hereditary skin disorders such as Fox-Poe Dise Syndrome or Hailey-Hailey disease; Keloid and hypertrophic scar reduction; Reduce pore size; Inflammatory conditions of the skin; Dermatological disorders including painful inflammatory conditions of the skin;

Includes pain associated with back pain (upper and lower back pain), fascia pain, tension headache, fibromyalgia, pain syndrome, myalgia, migraine, migraine, posttraumatic pain, pain not associated with muscle spasm and smooth muscle disorders Pain disorders;

Inflammatory disorders including pancreatitis, neurogenic inflammatory disorders (including gout, tendinitis, lupus, dermatomyositis and ankylosing spondylitis);

Hyperalgesia (including axillary hyperhidrosis, palmar hyperhidrosis and Frey's syndrome), hyperalgesic salivation, salivary hyperesthesia, sweat odor, mucus hypersecretion, hyperlactation, gonadal dysfunction; Excess sebum secretion;

Respiratory disorders including rhinitis (including allergic rhinitis), COPD, asthma and tuberculosis;

Hypertrophic disorders, including neurogenic tibialis hypertrophy with muscle enlargement, hypertrophic hypertrophy, acromegaly and myalgia;

Joint disorders including tennis elbow (or upper elbow inflammation), inflammation of the joints, arthropathy, osteoarthritis, rotator cuff tendinopathy of the shoulder, rheumatoid arthritis and wrist tunnel syndrome;

(Including hyperglycemia, hypercalcemia, hypothyroidism, hyperthyroidism, thyroid disorders (including Graves' disease, thyroiditis, Hashimoto thyroiditis, hyperthyroidism and hypothyroidism), hyperparathyroidism Endocrine and hypoparathyroidism), endocrine disorders such as Cushing's syndrome and obesity;

Autoimmune diseases such as systemic lupus erythematosus;

Proliferative diseases including adenocarcinoma tumors, prostate cancer and bone tumors;

Traumatic injuries, including sports injuries, muscle injuries, dry wounds and fractures; And

Veterinary uses (eg, immobilization of mammals, horse colic, animal imbalance or animal muscle spasms). The use of the pharmaceutical compositions according to the invention in cosmetics or aesthetics can also be used for the treatment of, for example, skin wrinkles, especially facial wrinkles such as facial frown lines, eye contour wrinkles, glabellar frown lines, Or prevention.

The pharmaceutical composition according to the present invention can also be used for the treatment of skin wrinkles, especially facial wrinkles such as facial wrinkles, eye contour wrinkles, non-wrinkled wrinkles, mouth wrinkles, Can be used for the treatment or prophylaxis of chin folds (peach d'orange, auxiliary chin), forehead wrinkles, "scratched skin" wrinkles, nose treatment or sleep lines .

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Harper Perennial, NY (1991)], as described in Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Provides a general descriptive general overview of many of the terms used in the present disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present disclosure. The numerical range includes a number defining the range. Unless otherwise indicated, each individual nucleic acid sequence is listed in 5 'to 3 ' orientation from left to right; Amino acid sequences are described as amino-carboxy-orientated from left to right respectively.

The headings provided herein do not limit the various aspects or embodiments of the disclosure which may be included by reference in the specification as a whole. Accordingly, the terms defined immediately below are defined in greater detail with reference to this specification as a whole.

Amino acids are referred to herein by the name of the amino acid, i. E., Three letter abbreviations or single letter abbreviations.

The term " amino acid sequence " as used herein is synonymous with the term " polypeptide " and / or the term " protein ". In some cases, the term " amino acid sequence " is synonymous with the term " peptide ". In some cases, the term " amino acid sequence " is synonymous with the term " enzyme ".

The term " protein " as used herein includes proteins, polypeptides, and peptides.

The terms " protein " and " polypeptide " are used interchangeably herein. In the present disclosure and claims, conventional 1-letter and 3-letter codes for amino acid residues may be used. The 3-letter code for amino acids is defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that polypeptides can be encoded by more than one nucleotide sequence due to the axial nature of the genetic code.

Other definitions of the term may appear throughout the specification. Before describing an exemplary embodiment in more detail, it is to be understood that this disclosure is not limited to the specific embodiments described, and, of course, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure should be limited only by the appended claims.

It is understood that, where a range of values is provided, values between each of up to one tenth of a unit of the lower limit between the upper and lower limits of the range are specifically disclosed, unless the context clearly indicates otherwise. Each smaller value range between any stated value or intervening value within the stated range and any other stated or intervening value within the stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may be independently included within the range or excluded, and each range in which any one, both, or both of the limits are included within a smaller range may also be included within the range Quot; is encompassed within the present disclosure subject to the specifically excluded limitations of < / RTI > Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the present disclosure.

It should be noted that the singular forms as used herein and in the appended claims include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to " Clostridial neurotoxin " includes a plurality of such candidate agents, and reference to " Clostridial neurotoxin " refers to one or more of the clathes known to those of ordinary skill in the art Tidium neurotoxin and its equivalents, and the like.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

The present invention will now be described, by way of example only, with reference to the following drawings and embodiments.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
Figure 1 shows the results of incubation of inactivated botulinum neurotoxin samples at 20 < 0 > C at a protein concentration of pH 8 and 0.55 mg / mL with recombinant pig trypsin (Roche) at final concentrations of 0.3 and 0.4 [ A full-length activated botulinum neurotoxin (endonegative) and an end-truncated activated botulinum neurotoxin heavy chain. Samples were separated for analysis by SDS-PAGE. Each SDS-PAGE sample was analyzed by density measurement.
Figure 2 shows that single-chain endo-negative botulinum neurotoxin E (0.55 mg / mL) at pH 8.0 is activated with small trypsin (Sigma-Aldrich) at various concentrations and incubated for 8 hours at 20 < The analysis by SDS-PAGE under reducing conditions is shown. ML trypsin; lane 6: 0.6 占 퐂 / mL trypsin; lane 1: molecular weight marker lane 2: -20 占 폚 control group lane 3: + 20 占 폚 control lane 4: 0.2 占 퐂 / Lane 7: 0.8 μg / mL trypsin; lane 8: 1.0 μg / mL trypsin).
Figure 3 shows the endo-negative botulinum neurotoxin E heavy chain (HC), light chain (LC) and truncated heavy chain (tHC) after activation with recombinant trypsin at pH 6.5, 7.0, 7.5 and 7.8 and incubation at 20 & (Neurotoxin concentration: 0.55 mg / mL, recombinant trypsin (Sigma-Aldrich) concentration: 1.5 μg / mL). Samples were analyzed by SDS-PAGE under reducing conditions by density measurement.
Figure 4 shows that the separation of the full-length, 2-chain botulinum neurotoxin E (endo-negative) from the truncated 2-chain botulinum neurotoxin was inhibited after activation with trypticin and separation using a ceramic hydroxyapatite type II chromatography column Can be achieved. The elution of the full-length 2-chain botulinum neurotoxin from the column and the truncated 2-chain botulinum neurotoxin was monitored by on-line A _{280 nm} reading and pooled selected fractions containing only the full-length 2-chain botulinum neurotoxin, And SDS-PAGE under non-reducing conditions.
Figure 5: Protein sequence of BoNT / A - Uniplot KB Accession No. P10845 (SEQ ID NO: 2).
Figure 6: Protein sequence of BoNT / B - Uniplot KB Accession No. P10844 (SEQ ID NO: 3).
Figure 7: Protein sequence of BoNT / C - Uniplot KB Accession No. P18640 (SEQ ID NO: 4).
Figure 8: Protein sequence of BoNT / D - Uniplot KB Accession No. P19321 (SEQ ID NO: 5).
Figure 9: Protein sequence of BoNT / E - Unipark ID UPI00000010A3 (SEQ ID NO: 6).
Figure 10: Protein sequence of BoNT / F - (SEQ ID NO: 7) of Uniplot KB Accession No. YP_001390123.
Figure 11: Protein sequence of BoNT / G - Uniplot KB Accession No. Q60393 (SEQ ID NO: 8).
12: Protein sequence of TeNT - Uniplot KB Accession No. P04958 (SEQ ID NO: 9).
Figure 13: Protein sequence of small trypsin (SEQ ID NO: 1).

Example

All publications mentioned in the above specification are incorporated herein by reference. Various modifications and alterations of the method and system of the invention described without departing from the scope and spirit of the invention will be apparent to those of ordinary skill in the art. While the invention has been described in connection with specific preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention, obvious to those of ordinary skill in biochemical and biotechnology or related art, are intended to fall within the scope of the following claims.

Example 1

Single-stranded botulinum neurotoxin E (endo-negative) samples at pH 8 and protein concentration of 0.55 mg / mL were incubated with recombinant pig trypsin (Roche) at final concentrations of 0.3 and 0.4 μg / mL at 20 ° C. For analysis by SDS-PAGE under reducing conditions, the samples were separated every 30 minutes up to 6 hours and then every 60 minutes until 9 hours. Each SDS-PAGE sample was analyzed by density measurement to determine the relative amounts of full-length 2-chain botulinum neurotoxin, truncated 2-chain botulinum neurotoxin heavy chain and single chain botulinum neurotoxin. Values for the full-length 2-chain botulinum neurotoxin and terminal truncated 2-botulinum neurotoxin were then plotted on the chart (Fig. 1). The truncated 2-chain botulinum neurotoxin occurred before contact with pig trypsin before complete activation of botulinum neurotoxin was achieved.

Example 2 - Activation by different concentrations of small trypsin

Single-stranded botulinum neurotoxin E (endo-negative) samples with a protein concentration of 0.55 mg / mL at pH 8.0 were incubated with small trypsin (Sigma-Aldrich) at 20, final concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 μg / ≪ / RTI > Samples were separated after 8 hours for analysis by SDS-PAGE under reducing conditions. The results shown in Figure 2 show that heavy chain termination was observed in each sample before complete activation was achieved.

Example 3 - Activation by Small Trypsin at Different pHs

Single-stranded botulinum neurotoxin E (endo-negative) samples with a protein concentration of 0.55 mg / mL at pH 6.5, 7.0, 7.5 and 7.8 were incubated with recombinant trypsin (Sigma-Aldrich) at a final concentration of 1.5 μg / Lt; / RTI > Samples were separated after 16 hours for analysis by SDS-PAGE under reducing conditions. Each SDS-PAGE sample was analyzed by density measurement to determine the relative amounts of truncated 2-chain botulinum neurotoxin heavy chain. The results are shown in FIG. 3 and Table 1. The truncated 2-chain botulinum neurotoxin formation occurs more readily at higher pH.

Table 1 - Percentage of truncated heavy chains at different pH (<LOD: below detection limit)

Example 4 - Purification of Activated Neurotoxin After Activation by Small Trypsin

26 mg of total protein containing endo-negative BoNT / E activated by incubation with 78.57 μg trypticin (small trypsin) at 20 ° C for 18 hours was applied to a 5 mL ceramic hydroxyapatite type II column. The column was washed with binding buffer (25 mM sodium phosphate, pH 6.5), followed by increasing the sodium phosphate concentration by a linear gradient over 35 column volumes using binding buffer and elution buffer (500 mM sodium phosphate pH 6.5) To collect 2.5 mL fractions. The eluates of the total length from the column, the 2-chain botulinum neurotoxin and the truncated 2-chain botulinum neurotoxin were monitored by on-line A280 nm reading and pooled selected fractions containing only the full-length 2-chain botulinum neurotoxin, And SDS-PAGE under non-reducing conditions (Figure 4).

Example 5 - Whole-body activated botulinum neurotoxin E formulation

Botulinum neurotoxin E inoculation. The coli cultures were prepared by dissolving the seed bank vial and inoculating into a shaking flask containing 100 mL of modified Terrific Broth (mTB). The flask was then incubated at 25 [deg.] C for 17 hours in a shaking incubator. Inoculum cultures were used to inoculate 5 shake flasks each containing 1 L of mTB. The cells were cultured in an incubator at 37 DEG C in an agitation incubator; The temperature of the culture was reduced to 16 캜; The culture was induced to a final concentration of 0.1 mM by the addition of isopropyl [beta] -D-1-thiogalactopyranoside (IPTG). Cells were harvested 20 hours after induction by tangential flow filtration (TFF) using a hollow fiber membrane. First, the culture was concentrated 10 times, followed by diafiltration with lysis buffer ₍₄ 100 mM sodium phosphate, 100 mM NaCl, 1.3 M ( NH) 2 SO 4, pH 7.8) 5 volumes.

The resulting cell paste slurry was homogenized by passing twice through a mechanical cell disruptor. The insoluble cell debris was sedimented by centrifugation and the supernatant was recovered and washed with butyl Sepharose 4 FF (100 mM NaCl, 100 mM NaCl, 1.25 M (NH ₄ ) ₂ SO ₄ , pH 7.8) &Lt; / RTI &gt; GE Lifesciences). The inactivated botulinum neurotoxin E was eluted from the column using a three step gradient with the following mixture of binding buffer and elution buffer (100 mM sodium phosphate, 100 mM NaCl, pH 7.8), and the product eluted in step 2:

Step 1 88% loading buffer; 12% elution buffer

Step 2 58% loading buffer; 42% elution buffer

Step 3 100% elution buffer

Subsequently, the material from step 2 was concentrated approximately two-fold by TFF with a hollow fiber membrane and then dialyzed against 10 volumes of 25 mM sodium phosphate pH 6.5 buffer. After dialysis filtration, any insoluble material in the retentate was precipitated by centrifugation, and the supernatant was applied to a Q Sepharose HP (Gly Life Scientific) packed column in a negative chromatographic step. A passing solution containing the inactivated botulinum neurotoxin was collected and the column was washed with 25 mM sodium phosphate pH 6.5 to maximize product recovery.

Then, the passed solution was incubated with 25 mM sodium phosphate and diluted to a total protein concentration of 0.5 mg / mL by the pH 6.5, / 7.27 USP units for 21 hours at room temperature mL recombinant bovine trypsin (trip binary ^®). The botulinum neurotoxin E, which was activated after incubation, was then applied to a ceramic hydroxyapatite Type II column, which was washed with binding buffer (25 mM sodium phosphate pH 6.5). The activated botulinum neurotoxin was eluted from the column by linear gradient using binding buffer and elution buffer (500 mM sodium phosphate pH 6.5).

Fractions containing the full-length, activated botulinum neurotoxin were pooled and applied to a benzamidine sequestrone FF (high-substituted) (gill life science) packed column in a negative chromatographic step. The passing solution containing the full length, activated botulinum neurotoxin was collected and the column was washed with loading buffer (110 mM sodium phosphate, pH 6.5) to maximize product recovery. The passing solution was dialyzed by TFF using a hollow fiber cartridge into a final storage buffer with 5 volumes of 25 mM sodium phosphate, 100 mM NaCl, pH 6.5.

                         SEQUENCE LISTING <110> IPSEN BIOPHARM LIMITED   <120> Production of activated clostridial neurotoxins <130> IBL 002-EP &Lt; 150 > EP 16177651.3 <151> 2016-07-01 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 246 <212> PRT <213> Bos taurus <400> 1 Met Lys Thr Phe Ile Phe Leu Ala Leu Leu Gly Ala Ala Val Ala Phe 1 5 10 15 Pro Val Asp Asp Asp Asp Lys Ile Val Gly Gly Tyr Thr Cys Gly Ala             20 25 30 Asn Thr Val Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe Cys         35 40 45 Gly Gly Ser Leu Ile Asn Ser Gln Trp Val Val Ser Ala Ala His Cys     50 55 60 Tyr Lys Ser Gly Ile Gln Val Arg Leu Gly Glu Asp Asn Ile Asn Val 65 70 75 80 Val Glu Gly Asn Glu Gln Phe Ile Ser Ala Ser Lys Ser Ile Val His                 85 90 95 Pro Ser Tyr Asn Ser Asn Thr Leu Asn Asn Asp Ile Met Leu Ile Lys             100 105 110 Leu Lys Ser Ala Ala Ser Leu Asn Ser Ser Val Ala Ser Ile Ser Leu         115 120 125 Pro Thr Ser Cys Ala Ser Ala Gly Thr Gln Cys Leu Ile Ser Gly Trp     130 135 140 Gly Asn Thr Lys Ser Ser Gly Thr Ser Tyr Pro Asp Val Leu Lys Cys 145 150 155 160 Leu Lys Ala Pro Ile Leu Ser Asp Ser Ser Cys Lys Ser Ala Tyr Pro                 165 170 175 Gly Gln Ile Thr Ser Asn Met Phe Cys Ala Gly Tyr Leu Glu Gly Gly             180 185 190 Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Ser Gly         195 200 205 Lys Leu Gln Gly Ile Val Ser Trp Gly Ser Gly Cys Ala Gln Lys Asn     210 215 220 Lys Pro Gly Val Tyr Thr Lys Val Cys Asn Tyr Val Ser Trp Ile Lys 225 230 235 240 Gln Thr Ile Ala Ser Asn                 245 <210> 2 <211> 1296 <212> PRT <213> Clostridium botulinum <400> 2 Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro             20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg         35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu     50 55 60 Ala Lys Gln Val Val Ser Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu                 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val             100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys         115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr     130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr                 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe             180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu         195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu     210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu                 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys             260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn         275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val     290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu                 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp             340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn         355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr     370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu                 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg             420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys         435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe     450 455 460 Ser Pro Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Glu Glu Glu Asn Ile Ser Leu                 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro             500 505 510 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu         515 520 525 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu     530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu 545 550 555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu                 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys             580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu         595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr     610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 640 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu                 645 650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala             660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys         675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu     690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys 705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu                 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn             740 745 750 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp         755 760 765 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile     770 775 780 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys                 805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly             820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp         835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser     850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875 880 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser                 885 890 895 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn             900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu         915 920 925 Lys Asn Ale Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser     930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn 945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val                 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu             980 985 990 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser         995 1000 1005 Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg     1010 1015 1020 Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln     1025 1030 1035 Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile     1040 1045 1050 Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp     1055 1060 1065 Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu     1070 1075 1080 Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys     1085 1090 1095 Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met     1100 1105 1110 Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val     1115 1120 1125 Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val     1130 1135 1140 Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr     1145 1150 1155 Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile     1160 1165 1170 Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn     1175 1180 1185 Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu     1190 1195 1200 Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser     1205 1210 1215 Gln Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn     1220 1225 1230 Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly     1235 1240 1245 Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala     1250 1255 1260 Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu     1265 1270 1275 Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu     1280 1285 1290 Arg Pro Leu     1295 <210> 3 <211> 1291 <212> PRT <213> Clostridium botulinum <400> 3 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asn Asn Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg             20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu         35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly     50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe                 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile             100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu         115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn     130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly                 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln             180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu         195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro     210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe                 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe             260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile         275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn     290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly                 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu             340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys         355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys     370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile                 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr             420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp         435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser     450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn 465 470 475 480 Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp                 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr             500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys         515 520 525 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln     530 535 540 Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp                 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Glu Ala Gly Leu Phe Ala Gly             580 585 590 Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser         595 600 605 Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile     610 615 620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro                 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile             660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys         675 680 685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp     690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr                 725 730 735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp             740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile         755 760 765 Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met     770 775 780 Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr                 805 810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu             820 825 830 Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile         835 840 845 Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile     850 855 860 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys                 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr             900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val         915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn     930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile                 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg             980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr         995 1000 1005 Asn Asn Asi Asn Ala Lys Ile Tyr Asle Asn Gly Lys Leu Glu     1010 1015 1020 Ser Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly     1025 1030 1035 Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe     1040 1045 1050 Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln     1055 1060 1065 Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr     1070 1075 1080 Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr     1085 1090 1095 Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys     1100 1105 1110 Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Ser Lys Tyr Asn     1115 1120 1125 Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly Glu     1130 1135 1140 Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp     1145 1150 1155 Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn     1160 1165 1170 Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys     1175 1180 1185 Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu     1190 1195 1200 Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr     1205 1210 1215 Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp     1220 1225 1230 Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile     1235 1240 1245 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr     1250 1255 1260 Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys     1265 1270 1275 Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu     1280 1285 1290 <210> 4 <211> 1291 <212> PRT <213> Clostridium botulinum <400> 4 Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn 1 5 10 15 Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu             20 25 30 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp         35 40 45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val     50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80 Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg                 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr             100 105 110 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp         115 120 125 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn     130 135 140 Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr                 165 170 175 Asn Asn Thr Phe Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile             180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp         195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile     210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile                 245 250 255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala             260 265 270 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr         275 280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu     290 295 300 Asn Ser Ile Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser                 325 330 335 Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn             340 345 350 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn         355 360 365 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr     370 375 380 Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395 400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser                 405 410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu             420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn         435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro     450 455 460 Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470 475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser                 485 490 495 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu             500 505 510 Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly         515 520 525 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu     530 535 540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu 545 550 555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala                 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly             580 585 590 Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Val Val Glu Asp         595 600 605 Phe Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp     610 615 620 Val Ser Ala Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val                 645 650 655 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly             660 665 670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys         675 680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser     690 695 700 Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe 705 710 715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly                 725 730 735 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser             740 745 750 Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu         755 760 765 Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg     770 775 780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn                 805 810 815 Leu Ile Asp Ser His Asn Ile Leu Val Gly Glu Val Asp Lys Leu             820 825 830 Lys Ala Lys Val Asn As Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile         835 840 845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr     850 855 860 Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu                 885 890 895 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly             900 905 910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn         915 920 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile     930 935 940 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe                 965 970 975 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn             980 985 990 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe         995 1000 1005 Phe Val Thr Val Thr Asn Met Met Gly Asn Met Lys Ile Tyr     1010 1015 1020 Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr     1025 1030 1035 Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile     1040 1045 1050 Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met     1055 1060 1065 Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys     1070 1075 1080 Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val     1085 1090 1095 Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr     1100 1105 1110 Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser     1115 1120 1125 Arg Gln Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn     1130 1135 1140 Glu Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn     1145 1150 1155 Asp Thr Arg Val Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr     1160 1165 1170 Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met     1175 1180 1185 Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu     1190 1195 1200 Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe Gln Ile     1205 1210 1215 Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe Lys     1220 1225 1230 Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly     1235 1240 1245 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His His Tyr     1250 1255 1260 Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu     1265 1270 1275 Ser Thr Ser Thr His Trp Gly Phe Val Ser Ser Glu     1280 1285 1290 <210> 5 <211> 1276 <212> PRT <213> Clostridium botulinum <400> 5 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr             20 25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu         35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro     50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg                 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val             100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp         115 120 125 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly     130 135 140 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly 145 150 155 160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly                 165 170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu             180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn         195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val     210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly 225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly                 245 250 255 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr             260 265 270 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln         275 280 285 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu     290 295 300 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn                 325 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser             340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn         355 360 365 Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe     370 375 380 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395 400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu                 405 410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu             420 425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser         435 440 445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys     450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480 Thr Asn Val Gln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile                 485 490 495 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu             500 505 510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val         515 520 525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr     530 535 540 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr                 565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly             580 585 590 Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn         595 600 605 Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile     610 615 620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640 Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu                 645 650 655 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe             660 665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn         675 680 685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met     690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn 705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala                 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn             740 745 750 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile         755 760 765 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val     770 775 780 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser                 805 810 815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val             820 825 830 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr         835 840 845 Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile     850 855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875 880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln                 885 890 895 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp             900 905 910 Lys Ile Ile Val Asn Leu Asn Asn Ile Leu Tyr Ser Ala Ile Tyr         915 920 925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr     930 935 940 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser 945 950 955 960 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln                 965 970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser             980 985 990 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr         995 1000 1005 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu     1010 1015 1020 Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp     1025 1030 1035 Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln     1040 1045 1050 Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser     1055 1060 1065 Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Glu Ile Leu Arg Asn     1070 1075 1080 Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu     1085 1090 1095 Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro     1100 1105 1110 Glu Ser Asn Val Leu Val Leu Val Gln Tyr Pro Asp Arg Ser Lys     1115 1120 1125 Leu Tyr Thr Gly Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys     1130 1135 1140 Asn Pro Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His     1145 1150 1155 Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp     1160 1165 1170 Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val     1175 1180 1185 Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr Gly Ile Gly     1190 1195 1200 Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr Cys Ser     1205 1210 1215 Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala Asp     1220 1225 1230 Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro     1235 1240 1245 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser     1250 1255 1260 Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu     1265 1270 1275 <210> 6 <211> 1252 <212> PRT <213> Clostridium botulinum <400> 6 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser             20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile         35 40 45 Gly Thr Thr Pro Gln Asp Phe His Pro Thr Ser Leu Lys Asn Gly     50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn                 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro             100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp         115 120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu     130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His                 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe             180 185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu         195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala     210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly                 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr             260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys         275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu     290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu                 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile             340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile         355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe     370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val                 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly             420 425 430 Glu Leu Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile         435 440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr     450 455 460 Glu Asn Asp Leu Asp Glu Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala                 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His             500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val         515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala     530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile                 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr             580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu         595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala     610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu 625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser                 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys             660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn         675 680 685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met     690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn                 725 730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser             740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser         755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu     770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr                 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp             820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys         835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp     850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser                 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr             900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn         915 920 925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg     930 935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn                 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe             980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn         995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile     1010 1015 1020 His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr     1025 1030 1035 Thr Arg Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu     1040 1045 1050 Leu Asp Glu Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn     1055 1060 1065 Thr Asn Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp     1070 1075 1080 Lys Glu Tyr Tyr Leu Leu Asn Val Leu Lys Pro Asn Asn Phe Ile     1085 1090 1095 Asp Arg Arg Lys Asp Ser Thr Leu Ser Ile Asn Asn Ile Arg Ser     1100 1105 1110 Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys Val Lys     1115 1120 1125 Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp Asn Leu Val Arg     1130 1135 1140 Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser Lys Thr His     1145 1150 1155 Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys Glu Lys     1160 1165 1170 Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val Val     1175 1180 1185 Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn     1190 1195 1200 Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr     1205 1210 1215 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr     1220 1225 1230 Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly     1235 1240 1245 Trp Gln Glu Lys     1250 <210> 7 <211> 1278 <212> PRT <213> Clostridium botulinum <400> 7 Met Pro Val Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys             20 25 30 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu         35 40 45 Arg Asn Thr Ile Gly Thr Asp Pro Ser Asp Phe Asp Pro Pro Ala Ser     50 55 60 Leu Glu Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys                 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Glu Val Leu Leu Gln Glu Ile Ser             100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Glu His Thr Pro Ile Asn Glu Phe         115 120 125 His Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Ser Ser Thr Asn     130 135 140 Val Lys Ser Ser Ile Ile Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 Asp Ile Phe Glu Asn Ser Ser Tyr Pro Val Arg Lys Leu Met Asp Ser                 165 170 175 Gly Gly Val Tyr Asp Pro Ser Asn Asp Gly Phe Gly Ser Ile Asn Ile             180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly         195 200 205 Gly Tyr Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser     210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 Gly Val Thr Tyr Lys Glu Thr Ile Lys Val Lys Gln Ala Pro Leu Met                 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly             260 265 270 Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn         275 280 285 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Arg Val     290 295 300 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val                 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr             340 345 350 Glu Ile Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr         355 360 365 Phe Ile Lys Tyr Gly Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp     370 375 380 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400 Asn Arg Gly Gln Asn Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile                 405 410 415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val             420 425 430 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val         435 440 445 Asn Asn Arg Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu     450 455 460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser                 485 490 495 Glu Thr Ile Pro Gln Ile Ser Asn Gln Thr Leu Asn Thr Leu Val Gln             500 505 510 Asp Asp Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile         515 520 525 Glu Glu His Asn Val Val Asp Leu Asn Val Phe Phe Tyr Leu His Ala     530 535 540 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560 Asp Thr Ala Leu Ser Glu Glu Ser Gln Val Tyr Thr Phe Ser Ser                 565 570 575 Glu Phe Ile Asn Thr Ile Asn Lys Pro Val His Ala Ala Leu Phe Ile             580 585 590 Ser Trp Ile Asn Gln Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln         595 600 605 Lys Ser Thr Phe Asp Lys Ile Ala Asp Ile Ser Leu Val Val Pro Tyr     610 615 620 Val Gly Leu Ala Leu Asn Ile Gly Asn Glu Val Gln Lys Glu Asn Phe 625 630 635 640 Lys Glu Ala Phe Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val                 645 650 655 Pro Glu Leu Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe             660 665 670 Ile Gly Ser Ser Glu Asn Lys Asn Lys Ile Ile Lys Ala Ile Asn Asn         675 680 685 Ser Leu Met Glu Arg Glu Thr Lys Trp Lys Glu Ile Tyr Ser Trp Ile     690 695 700 Val Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys 705 710 715 720 Glu Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr                 725 730 735 Val Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Arg Asn Arg             740 745 750 Leu Glu Ser Glu Tyr Asn Ile Asn Asn Ile Arg Glu Glu Leu Asn Lys         755 760 765 Lys Val Ser Leu Ala Met Glu Asn Ile Glu Arg Phe Ile Thr Glu Ser     770 775 780 Ser Ile Phe Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Ser Lys 785 790 795 800 Leu Arg Glu Tyr Asp Glu Gly Val Lys Glu Tyr Leu Leu Asp Tyr Ile                 805 810 815 Ser Glu His Arg Ser Ile Leu Gly Asn Ser Val Gln Glu Leu Asn Asp             820 825 830 Leu Val Thr Ser Thr Leu Asn Asn Ser Ile Pro Phe Glu Leu Ser Ser         835 840 845 Tyr Thr Asn Asp Lys Ile Leu Ile Leu Tyr Phe Asn Lys Leu Tyr Lys     850 855 860 Lys Ile Lys Asp Asn Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys 865 870 875 880 Phe Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asp                 885 890 895 Val Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Ser Ser             900 905 910 Lys Pro Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn         915 920 925 Gly Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys     930 935 940 Tyr Phe Asn Lys Val Asn Leu Asn Asn Glu Tyr Thr Ile Ile Asp Cys 945 950 955 960 Ile Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr Asn Lys                 965 970 975 Ile Ile Trp Thr Leu Gln Asp Thr Ala Gly Asn Asn Gln Lys Leu Val             980 985 990 Phe Asn Tyr Thr Gln Met Ile Ser Ile Ser Asp Tyr Ile Asn Lys Trp         995 1000 1005 Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser Arg Ile     1010 1015 1020 Tyr Ile Asn Gly Asn Leu Ile Asp Glu Lys Ser Ile Ser Asn Leu     1025 1030 1035 Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Gly     1040 1045 1050 Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe     1055 1060 1065 Asp Thr Glu Leu Gly Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asp     1070 1075 1080 Glu Pro Asp Pro Ser Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu     1085 1090 1095 Leu Tyr Asn Lys Arg Tyr Tyr Leu Leu Asn Leu Leu Arg Thr Asp     1100 1105 1110 Lys Ser Ile Thr Gln Asn Ser Asn Phe Leu Asn Ile Asn Gln Gln     1115 1120 1125 Arg Gly Val Tyr Gln Lys Pro Asn Ile Phe Ser Asn Thr Arg Leu     1130 1135 1140 Tyr Thr Gly Val Glu Val Ile Ile Arg Lys Asn Gly Ser Thr Asp     1145 1150 1155 Ile Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr     1160 1165 1170 Ile Asn Val Val Asp Arg Asp Val Glu Tyr Arg Leu Tyr Ala Asp     1175 1180 1185 Ile Ser Ile Ala Lys Pro Glu Lys Ile Ile Lys Leu Ile Arg Thr     1190 1195 1200 Ser Asn Ser Asn Asn Ser Leu Gly Gln Ile Ile Val Met Asp Ser     1205 1210 1215 Ile Gly Asn Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Gly     1220 1225 1230 Asn Ile Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser     1235 1240 1245 Ser Trp Tyr Asn Asn Ile Arg Lys Asn Thr Ser Ser Asn Gly     1250 1255 1260 Cys Phe Trp Ser Phe Ile Ser Lys Glu His Gly Trp Gln Glu Asn     1265 1270 1275 <210> 8 <211> 1297 <212> PRT <213> Clostridium botulinum <220> <221> misc_feature <222> (7) (7) <223> Xaa can be any naturally occurring amino acid <400> 8 Met Pro Val Asn Ile Lys Xaa Phe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15 Asp Asp Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr             20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu         35 40 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly     50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe                 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile             100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys         115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln     130 135 140 Pro Gly Ala Glu Asp Gly Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile                 165 170 175 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met             180 185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu         195 200 205 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro     210 215 220 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe                 245 250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe             260 265 270 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile         275 280 285 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn     290 295 300 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Sel 305 310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys                 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met             340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr         355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys     370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn                 405 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg             420 425 430 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu         435 440 445 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys     450 455 460 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile                 485 490 495 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr             500 505 510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys         515 520 525 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu     530 535 540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu 545 550 555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr                 565 570 575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala             580 585 590 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser         595 600 605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Ser Ser Ile     610 615 620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu                 645 650 655 Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr             660 665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser         675 680 685 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu     690 695 700 Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile 705 710 715 720 Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu                 725 730 735 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met             740 745 750 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser         755 760 765 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser     770 775 780 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu 785 790 795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp                 805 810 815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys             820 825 830 Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr         835 840 845 Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn     850 855 860 Ile Ser Ser Asn Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 Ile Asp Ser Ser Gly Ser Ser Val Ser Ser Val Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser                 885 890 895 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu             900 905 910 Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser         915 920 925 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr     930 935 940 Asn Asn Asn Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn                 965 970 975 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile             980 985 990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys         995 1000 1005 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn     1010 1015 1020 Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn     1025 1030 1035 Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile     1040 1045 1050 Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn     1055 1060 1065 Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr     1070 1075 1080 Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn     1085 1090 1095 Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly Met     1100 1105 1110 Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu     1115 1120 1125 Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln     1130 1135 1140 Asn Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn     1145 1150 1155 Ser Arg Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr     1160 1165 1170 Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg Val     1175 1180 1185 Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln Leu Phe Leu     1190 1195 1200 Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu Gln Ile     1205 1210 1215 Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu Cys     1220 1225 1230 Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe     1235 1240 1245 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe     1250 1255 1260 Cys Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn     1265 1270 1275 Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu     1280 1285 1290 Gly Trp Thr Glu     1295 <210> 9 <211> 1315 <212> PRT <213> Clostridium tetani <400> 9 Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn 1 5 10 15 Asp Thr Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp Ile             20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Val Pro Glu         35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe Asn Pro Pro Ser Ser     50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr 65 70 75 80 Asp Ser Asp Lys Asp Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn                 85 90 95 Arg Ile Lys Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile             100 105 110 Asn Ale Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe         115 120 125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro     130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Gly Val Arg Gly Ile Val Leu                 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys Arg Asp Gly Phe Gly Ser             180 185 190 Ile Met Gln Met Ala Phe Cys Pro Glu Tyr Val Pro Thr Phe Asp Asn         195 200 205 Val Ile Glu Asn Ile Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe     210 215 220 Gln Asp Pro Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His 225 230 235 240 Gly Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys                 245 250 255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu             260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys         275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn     290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp Ile Asp 305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln Phe Asp Lys Asp Ser                 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn             340 345 350 Ser Ile Met Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn         355 360 365 Ile Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys     370 375 380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe 385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met                 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu Val             420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro Thr Asn Ile         435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu Thr Asp Leu Gly Gly     450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 465 470 475 480 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr                 485 490 495 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile             500 505 510 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg         515 520 525 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser     530 535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile                 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile             580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln         595 600 605 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr     610 615 620 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ser Ser Val Ser Ser 625 630 635 640 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly                 645 650 655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu             660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu         675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile     690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 705 710 715 720 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys                 725 730 735 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile             740 745 750 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys         755 760 765 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu     770 775 780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln                 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile             820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu         835 840 845 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser     850 855 860 Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile 865 870 875 880 Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile                 885 890 895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala             900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn         915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn     930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Val Lys 945 950 955 960 Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile                 965 970 975 Ile Ser Ser Lys Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser             980 985 990 Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala         995 1000 1005 Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe     1010 1015 1020 Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn     1025 1030 1035 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met     1040 1045 1050 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn     1055 1060 1065 Asn Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr     1070 1075 1080 Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro     1085 1090 1095 Lys Glu Ile Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe     1100 1105 1110 Leu Arg Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr     1115 1120 1125 Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val Gln Leu Lys     1130 1135 1140 Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser Tyr Thr     1145 1150 1155 Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly Leu     1160 1165 1170 Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser     1175 1180 1185 Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn     1190 1195 1200 Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe     1205 1210 1215 Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly     1220 1225 1230 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu     1235 1240 1245 Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala     1250 1255 1260 Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp     1265 1270 1275 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His     1280 1285 1290 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr     1295 1300 1305 Asp Glu Gly Trp Thr Asn Asp     1310 1315 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> E activation loop <400> 10 Cys Lys Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile Cys 1 5 10 15

Claims (17)

Contacting the single-stranded Clostridium neurotoxin with an activating enzyme until at least 90% of the single-stranded Clostridium neurotoxin is converted to a 2-chain Clostridium neurotoxin, wherein the activated Clostridium neurotoxin &Lt; / RTI &gt; 2. The method of claim 1, wherein the activating enzyme is trypsin. 3. The method of claim 2 wherein the trypsin is trypticin and the small trypsin has an amino acid sequence that is at least 90% identical to SEQ ID NO: 4. The method of claim 3, wherein the small trypsin is selected from native trypsin and recombinant small trypsin obtained from a bovine pancreas. 5. The method of claim 3 or 4, wherein said step of contacting said single-stranded Clostridial neurotoxin with small trypsin is carried out at a pH of from 5 to 7.5, preferably from 6 to 7, for example at a pH of about 6.5 Lt; / RTI &gt; 6. The method of claim 5 wherein contacting the single stranded Clostridium neurotoxin with the small trypsin is performed at room temperature at a pH of 6 to 7 for a duration of 15 to 25 hours and the concentration of the small trypsin is Clostridium And 0.5 to 3 [mu] g per mg neurotoxin. 7. The method of any one of claims 1 to 6, further comprising the step of removing the truncated Clostridial neurotoxin. 8. The method of claim 7, wherein the step of removing truncated Clostridial neurotoxin comprises contacting the activated Clostridial neurotoxin with a mixed mode chromatography resin. 9. The method of any one of claims 1 to 8 wherein the Clostridial neurotoxin is selected from the group consisting of BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / &Lt; / RTI &gt; 10. A method according to any one of claims 1 to 9, wherein the Clostridial neurotoxin comprises a modified botulinum neurotoxin as compared to a wild-type botulinum neurotoxin, for example a mutated Clostridial neurotoxin, a chimeric Clostridium neurotoxin, Toxin or a retargeted Clostridial neurotoxin. 11. The method according to any one of claims 1 to 10, wherein the Clostridial neurotoxin comprises a BoNT / E activation loop. 12. The method according to any one of claims 1 to 11, wherein the Clostridial neurotoxin is BoNT / E. 13. The method according to any one of claims 1 to 12, wherein the single-stranded Clostridial neurotoxin encodes the single-stranded Clostridial neurotoxin in a heterologous host cell, Lt; RTI ID = 0.0 &gt; E. coli. &Lt; / RTI &gt; 13. An active two-chain Clostridium neurotoxin obtainable by the method of any one of claims 1 to 13. 15. A pharmaceutical composition comprising an active 2-chain Clostridium neurotoxin according to claim 14, wherein the composition is essentially free of single-stranded Clostridium neurotoxin. 16. Use according to claim 15 for the manufacture of a medicament for use in therapy, for example for the treatment of ophthalmologic disorders, movement disorders, otorhinolaryngological disorders, gastrointestinal disorders, genitourinary disorders, dermatological disorders, pain disorders, inflammatory disorders, secretory disorders, Disorders, endocrine disorders, autoimmune diseases, proliferative diseases, treatment of traumatic injuries, veterinary uses. Use of a pharmaceutical composition according to claim 15 in cosmetics or aesthetics.

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